The Cas9-gRNA ribonucleoprotein complex-mediated editing of pyrG in Ganoderma lucidum and unexpected insertion of contaminated DNA fragments

Gene editing is a promising alternative to traditional breeding for the generation of new mushroom strains. However, the current approach frequently uses Cas9-plasmid DNA to facilitate mushroom gene editing, which can leave residual foreign DNA in the chromosomal DNA raising concerns regarding genetically modified organisms. In this study, we successfully edited pyrG of Ganoderma lucidum using a preassembled Cas9-gRNA ribonucleoprotein complex, which primarily induced a double-strand break (DSB) at the fourth position prior to the protospacer adjacent motif. Of the 66 edited transformants, 42 had deletions ranging from a single base to large deletions of up to 796 bp, with 30 being a single base deletion. Interestingly, the remaining 24 contained inserted sequences with variable sizes at the DSB site that originated from the fragmented host mitochondrial DNA, E. coli chromosomal DNA, and the Cas9 expression vector DNA. The latter two were thought to be contaminated DNAs that were not removed during the purification process of the Cas9 protein. Despite this unexpected finding, the study demonstrated that editing G. lucidum genes using the Cas9-gRNA complex is achievable with comparable efficiency to the plasmid-mediated editing system.

www.nature.com/scientificreports/ Gene editing in mushrooms has emerged as a promising molecular breeding tool to perform targeted manipulation of genes related to specific traits. The announcement of the gene-edited A. bisporus to bypass US GMO regulations in 2016 has been a significant driver of this approach 10 . Several species of mushrooms, including Ceriporiopsis subvermispora 11 , Coprinopsis cinerea 12 , Cordyceps militaris [13][14][15] , Flammulina filiformis 16 , G. lucidum [17][18][19][20] , L. edodes 21 , P. ostreatus [22][23][24] , and P. eryngii 25 , have been subjected to gene editing through the introduction of plasmid DNA carrying Cas9 and guide RNA (gRNA). The plasmid DNA here is either integrated into the chromosomal DNA or diluted out along with mycelial cell division due to the absence of autonomously replicative sequences. ATMT is another way to introduce the Cas9 and gRNA genes. Zhang et al. 26 achieved the disruption of pyrG by chromosome-integrated Cas9 and gRNA and subsequent disruption of lacA or mnp9 through additional integration of gRNA targeting lacA or mnp9 using ATMT.
Another intriguing approach to implementing gene editing is by directly introducing the Cas9-gRNA ribonucleoprotein complex (RNP) into protoplasts. The major advantage of this approach is that it does not involve chromosomal integration of Cas9/gRNA, enabling marker-free editing. In Vonk et al. 's 27 study, RNP was utilized to create a targeted DSB in the hom2 gene of Schizophyllum commune. Selection of the hom2-edited cells was facilitated by cotransformation of a DNA fragment containing a selective marker gene flanked by homologous sequences of hom2, which was integrated into the hom2 gene through HR upon the creation of a DSB. A similar strategy was employed to edit cre1 of Coprinopsis cinerea 28 and pyrG of P. ostreatus 29 .
G. lucidum is commonly found in Asia 30 and has gained popularity due to its active compounds, such as ganoderic acids, which have been demonstrated to effectively treat various diseases, such as cancer, diabetes, and hypertension; as a result, G. lucidum is a valuable source of medicinal substances [31][32][33] . Qin et al. 17 reported the first gene-edited G. lucidum by plasmid carrying the Cas9 gene with in vitro transcribed gRNA targeting pyrG (ura3), which encodes orotidine 5-phosphate decarboxylase and has been a frequent target in gene editing due to its disruption enables positive selection against 5-fluoroorotic acid (5-FOA). The pyrG-edited strain was further subjected to modification of cyp5150l8, which is involved in the biosynthesis of ganoderic acids 18 . Similar plasmid-mediated editing was performed to target pyrG and GL17624 19 and ku80 and pyrG 20 . These plasmidbased editing systems have their own issues in general use, in which several factors should be considered, such as codon optimization of the Cas9 gene, promoter selection for the expression of Cas9 and gRNA, use of selective markers, and integration of plasmid DNA fragments into chromosomal DNA.
In this regard, we attempted to develop a gene editing system in G. lucidum using purified Cas9 protein assembled together with in vitro transcribed gRNA. We found that the Cas9-gRNA RNP could effectively edit pyrG with a broad range of deletions through NHEJ repair. We also found that the DSB was repaired by insertion of random DNA fragments, originating from the transformation reaction mixture and from the mitochondrial DNA (mtDNA) fragments.

Results
The Cas9 protein expressed in E. coli was purified by Ni-NTA column chromatography (Fig. 1a). SDS-PAGE analysis of the eluted fraction (Fig. 1a, Lane 4) showed that Cas9 was the major protein, while minor contaminants with molecular weights of less than 75 kDa were also present. These minor proteins did not affect the Cas9 cleavage activity (Fig. 1c) and were thought to be E. coli proteins non-specifically bound to Ni-NTA column during the purification process. The gRNA was synthesized by in vitro transcription as depicted in Fig. 1b. Next, we examined cleavage of pyrG by gRNA-guided Cas9 activity. The pyrG DNA (1065 bp) was cut in a Cas9 protein concentration-dependent manner (Fig. 1c). However, the cleavage efficiency varied depending on the gRNA used. For example, Cas9 with gRNA1, which targets the first exon in pyrG, cut most of the target sequence into two fragments with sizes of 826 bp and 239 bp. pyrG band density analysis revealed that Cas9 complexed with gRNA1, gRNA2, and gRNA3 cleaved 79%, 37%, and 51% of input pyrG DNA, respectively (Fig. 1d).
To investigate the effect of gRNA on the transformation efficiency, the three Cas9-gRNA complexes were assembled by incubating the purified Cas9 protein with the in vitro-synthesized gRNA1, gRNA2, or gRNA3. The Cas9-gRNA complex was independently introduced to the protoplasts by PEG-mediated transformation. The transformants were selected based on their ability to survive or die on YMGUU + FOA medium. Only transformants with disrupted pyrG could survive in the presence of 5-FOA, as the orotidine 5'-phosphate (OMP) decarboxylase encoded by pyrG converts 5-FOA into lethal 5-fluorouracil. After conducting three independent transformation experiments, we retrieved a total of 199, 3, and 24 isolates from gRNA1, gRNA2, and gRNA3, respectively (Fig. 2a). The fast-growing isolates were further selected on a new YMGUU + FOA to obtain 66 transformants from gRNA1 and one from gRNA3. The growth characteristics of some selected transformants are shown in Fig. 2b, in which the transformants grew well on YMGUU + FOA, while the wild-type strain could not. The transformants were less viable on YMG medium, which lacked uracil and uridine, because of the disrupted pyrG.
We subsequently investigated the pyrG gene in the transformants selected from gRNA1 using the in vitro cleavage assay. The pyrG DNA in the transformants was amplified by PCR and reacted with the Cas9 protein and gRNA1. While the wild-type pyrG was cut by two pieces through the activity of the Cas9-gRNA1 complex (Fig. 2c), all pyrGs from the transformants were resistant to Cas9-gRNA1, indicating modifications in the sequence region corresponding to crRNA. Moreover, there were noticeable changes in the length of pyrG from the transformants N21, N39, N22, and N56. pyrG from N21 was reduced to 674 bp, whereas N39, N22, and N56 carried noticeably larger pyrG than that of the wild type (Fig. 2c). pyrGs in the 66 transformants were amplified by a primer set (Supplementary Table S1) and were subjected to sequence determination. Investigation of the obtained sequences revealed that all 66 pyrGs were modified by the Cas9-gRNA1 complex, which potentially led to the inactivation of OMP decarboxylase. Forty-two transformants were found to contain deletions in the pyrG region (Fig. 3), whereas twenty-four contained insertions with variable lengths and origins (Fig. 4a) www.nature.com/scientificreports/ the deletions, thirty were single base deletions at the fourth position before the PAM sequence ( Fig. 3, N2 as a representative transformant). The next most frequent deletion was a two-base deletion at the fourth position before PAM, found in five transformants, including N30. The transformant N12 carried a three-base deletion at the second position before PAM, which caused amino acid residues isoleucine 52 and valine 53 to change into methionine 52 (Fig. 3a, boxed). This change effectively disrupted the OMP decarboxylase activity, as shown by survival on YMGUU + FOA and death on YMG (Fig. 2b). Large deletions were also found in the six transformants ( Fig. 3b, Supplementary Fig. S1). The transformants N3 and N35 showed 152-bp and 99-bp deletions, respectively, in exon 1 of pyrG. The deleted regions included the gRNA1 target sequence. N32 showed a 337-bp deletion, ranging from the fourth position before PAM to the upstream 179-bp region. The N9 transformants showed a deletion of 439 bp, including the upstream 84 bp, exons 1 and 2, and a part of the second intron sequence. The second largest deletion was found in N21, in which a 674-bp deletion occurred from 29 bp after the start codon to a significant part of exon 3. N15 showed the largest deletion (796 bp), ranging from 92 bp upstream to half of exon 3. Interestingly, in this transformant, the deleted sequence was replaced by a 50-bp DNA fragment, which belonged to a noncoding sequence region of G. lucidum mtDNA. Sequence analysis of pyrGs revealed that twenty-four transformants contained insertions of different sizes at the gRNA region of exon 1 (Fig. 4a, Supplementary Table S2). The insertions primarily occurred at the 4th base from the PAM sequence. The smallest insertion was a single base insertion in N23, whereas the largest was 420 bp found in N42. Detailed sequence analysis revealed that the inserted sequences come from the following different origins: pTrc99-Cas9 plasmid DNA, E. coli chromosomal DNA, and the mtDNA of G. lucidum (depicted in Fig. 4b). Six transformants, including N4, N27, N29, N37, N40, and N61, carried inserted sequences originating from the vector DNA pTrc99-Cas9, which was used to produce the Cas9 protein (Fig. 4a, Supplementary  Fig. S2). DNA fragments from E. coli, which was the host for pTrc99-Cas9, were also found to be inserted into 8 transformants (Fig. 4a). These results indicate that the fragmented DNAs generated during the preparation of the Cas9 protein remained as contaminants in the protein solution. Then, these fragments were incorporated into the target sequence when the DSB occurred due to Cas9-gRNA1 activity. The other type of inserted sequence was from mtDNA of G. lucidum (Fig. 4a, Supplementary Fig. S2). Nine transformants carried mtDNA fragments, of www.nature.com/scientificreports/ which N10 and N11 had the same insertion, which originated from the cox3 gene, and N34, N66, and N74 possessed the same inserted sequence, which originated from cox1. These mtDNA fragments conceivably originated from disrupted mitochondria during the transformation process. The repeated occurrence of the two mtDNA fragments in the five transformants (N10, N11, N34, N66, and N74) may suggest nonrandom fragmentation of mtDNA ( Supplementary Fig. S2). In contrast, the inserted sequences were randomly distributed to the vector DNA and E. coli chromosomal DNA. The transformant generated from gRNA3 contained the inserted sequence originating from E. coli chromosomal DNA (Supplementary Table S2).

Discussion
Over the past few years, significant advancements have been made in the field of mushroom gene editing, particularly through successful transformations using plasmid DNA carrying the cas9 gene into protoplasts. However, for the practical use of this plasmid-based editing system, various factors must be carefully considered. For example, the appropriate promoters for the expression of Cas9 and gRNA must be chosen, the codons of Cas9 must be optimized to ensure efficient translation into protein, which requires a selective marker for cytoplasmic maintenance, and fragmented plasmid DNA may be randomly integrated into chromosomal DNA. In this regard, directly introducing the Cas9-gRNA RNP complex to protoplasts has been attempted for gene editing in mushrooms, such as S. commune 27 , C. cinerea 28 , and P. ostreatus 29,34 .   www.nature.com/scientificreports/ In this study, we found that the preassembled RNP was effective in disrupting pyrG in G. lucidum by inducing a double-strand break at the 4th base before PAM, similar to the results obtained for F. filiformis 16 and P. ostreatus 22 . The efficiency of transformation was found to be highly dependent on the gRNA used and was directly proportional to the in vitro cleavage activity of the Cas9-gRNA complex. Out of the three gRNAs tested, only gRNA1, which exhibited the best cleavage activity in the in vitro cleavage assay (Fig. 1c), consistently produced a reasonable number of transformants, averaging 22 per 10 7 protoplasts (as shown in Fig. 2a). Compared to the plasmid method, which exhibited an average efficiency of pyrG disruption of 16 per 10 7 protoplasts 19 , our RNPbased transformation was found to be comparable.
Disruption of the pyrG gene was observed to result from deletions and insertions around the DSB site. Out of the 42 deleted transformants, 30, 5, and 1 exhibited single base, two base, and three base deletions, respectively, while the remaining six transformants showed larger deletions ranging from 99 to 796 bp, which may have occurred during the repair process via NHEJ. A more intriguing observation from the repair process was the insertion of DNA fragments from various sources. Specifically, 24 transformants were found to contain inserted sequences ranging in size from a single base to 420 bp; based on sequence analysis, these sequences originated from the following sources: contaminated DNA fragments in the purified Cas9 protein and mtDNA of host cells. The DNA fragments contaminating the purified Cas9 proteins originated from the expression vector DNA and the E. coli chromosomal DNA generated during the disruption of the Cas9-overexpressing E. coli cells by sonication. Additionally, mtDNA fragments may have been generated during the transformation process. As mushrooms may contain a substantial amount of mtDNA (similar to yeast cells, which can have 50-200 mitochondria 35 ), the high osmotic and physicochemical stresses during protoplast generation and PEG-mediated transformation could have accelerated mtDNA degradation by nuclease activities 36 , resulting in the accumulation and insertion of degraded mtDNA fragments. Notably, this study found that certain transformants contained identical insertions in N10 and N11 and in N34, N66, and N74 (Fig. 4a), which suggests the involvement of a specific endonuclease in mtDNA degradation. Lastly, the random insertion of DNA fragments into the DSB site suggests that insertion of a specific DNA fragment can be facilitated when the RNP is transformed together with a DNA fragment with a specific function without using HR. This type of RNP-mediated gene editing and subsequent positive selection was employed in the double-gene targeting of P. ostreatus, in which both pyrG and fcy1 (encoding cytosine deaminase) were disrupted by a single transformation of Cas9/pyrGsg1 and Cas9/fcy1sg2 RNPs and selected against 5-FOA and 5-fluorocytosine (5-FC) 34 . Deamination of 5-FC by fcy1 produces 5-FU which is the same toxic product produced by pyrG on 5-FOA.
Although the use of RNPs enables marker-free editing, it has limitations regarding the selection process for modified organisms. These limitations arise unless the disruption of the target gene results in a discernible phenotype, such as the survival of the edited organism under 5-FOA selection due to pyrG disruption. Therefore, RNP-mediated gene editing is constrained when it comes to targeting arbitrary genes unless an effective selection strategy is in place.

Materials and Methods
Strain and culture conditions. A monokaryotic strain of G. lucidum was isolated by dedikaryotization of a dikaryotic strain (GL3315), which was isolated from the Sorak Mountains located in the northwestern part of Korea. The isolated strain was cultured at 28 °C on YPD medium containing yeast extract (5 g/L) and potato dextrose agar (24 g/L) or on minimal medium composed of asparagine (2 g/L), MgSO 4 (0.12 g/L), dextrose (5 g/L), potato starch (5 g/L), and thiamine (1 µg/L).
Design and synthesis of gRNA. Three crRNAs targeting pyrG of G. lucidum (GenBank ID: JQ406674.1) were designed using CRISPOR (http:// crisp or. tefor. net/). For the synthesis of gRNA, the T7 promoter (P T7 ), crRNA, and tracrRNA were connected by PCR with primer sets (Supplementary Table S1) using pHAtC-LeCas9 21 as template DNA. The resulting DNA fragments were subjected to in vitro transcription using an in vitro RNA synthesis kit (NEB, Ipswich, MA). The gRNAs were purified using an RNA purification kit (Invitrogen, Waltham, MA).

Purification of Cas9 protein. The Cas9 expression vector (pTrc-Cas9) was obtained from Prof. Hojin
Ryu at Chungbuk National University. E. coli carrying pTrc-Cas9 was grown in LB broth containing ampicillin (50 μg/mL) at 37 ℃. Expression of Cas9 was induced by 0.1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) when the OD600 reached 0.6. The induced broth was further incubated for 16 h. The cells were harvested by centrifugation at 10,000×g for 20 min. The collected cells were disrupted by sonication in a 50 mM sodium phosphate buffer containing 0.1 M NaCl (pH 7.5). The disrupted solution was centrifuged at 10,000×g for 30 min (4 °C). The supernatant was collected and applied to a Ni-NTA column (10 mL) equilibrated with 50 mM sodium phosphate buffer containing 0.1 M NaCl (pH 7.5). The column was washed with 100 ml of equilibration buffer supplemented with 20 mM imidazole. The bound Cas9 protein was eluted using 1 M imidazole in the equilibration buffer. The eluate was dialyzed against 50 mM sodium phosphate buffer for 12 h at 4 °C to obtain the purified Cas9 protein. A Bradford assay was performed to determine the protein concentration.
Evaluating the gRNA performance by an in vitro cleavage assay. The efficiency of the gRNAs in the editing of pyrG was evaluated by an in vitro cleavage assay. Purified Cas9 at different concentrations (10, 100, and 1000 ng) was incubated with the three gRNAs (500 ng) in the presence of 200 ng pyrG DNA fragment, which was amplified by PCR using chromosomal DNA extracted from G. lucidum. The cleavage reaction was performed in reaction buffer (20 μL) consisting of 50 mM Tris-HCl, 0.1 M NaCl, 10 mM MgCl 2 , and 1 mM DTT (pH 7.9) at 37 °C for 10 min. The reaction mixture was analyzed by 1.5% agarose gel electrophoresis. The

Data availability
All data generated or analysed during this study are included in this published article and its supplementary information files.